Differential expression of genes in the WT and hupB KO strain of Mycobacterium tuberculosis H37Rv upon iron limitation. Mycobacterium tuberculosis

HupB is a 28 kDa in Mycobacterium tuberculosis that is co-expressed with the siderophores mycobactin and carboxymycobactin upon iron limitation. High levels of all the three components are seen in low iron (LI; 0.02 µg Fe / mL) organisms, with negligible expression in high iron organisms (HI; 8 µg Fe / mL). We generated a hupB knock out mutant of M. tuberculosis (H37Rv ∆ hupB) and studied the differential expression of genes upon iron limitation in the WT H37Rv and the mutant. The RNA transcripts of the WT H37Rv, grown under high and low iron conditions of growth were isolated and subjected to microarray analysis to identify the iron-regulated genes and second, the differential expression of genes in iron-limited H37Rv ∆ hupB vs iron-limited WT H37Rv was analysed. Microarray analysis was done commercially by Genotypic Technology (Bangalore, India), an authorised service provider for Agilent Technologies. The study revealed the up-regulation of all the mbt genes of the mycobactin biosynthetic machinery in LI - H37Rv and several other reported iron-regulated genes. The salient feature of this study is the failure of LI - H37Rv ∆ hupB to show any up-regulation of the mbt genes as compared to LI - H37Rv. Among several other genes influenced by HupB, the mutant strain showed low levels of mmpL5 and mmpS5 transcripts, whose expressed products are reported to be associated with siderophore transport and biosynthesis. Overall design: One-color experiment,Organism: Mycobacterium tuberculosis ,Custom Mycobacterium tuberculosis 8x15k Array designed byGenotypic Technology Private Limited (AMADID: 20181), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)

HupB是结核分枝杆菌（Mycobacterium tuberculosis）中一种分子量为28 kDa的蛋白质，在铁限制培养条件下，会与铁载体分枝杆菌素（mycobactin）及羧基分枝杆菌素（carboxymycobactin）共表达。低铁（LI；0.02 μg Fe/mL）培养的菌体中，该蛋白与两种铁载体的表达量均处于较高水平，而高铁（HI；8 μg Fe/mL）培养的菌体中几乎检测不到其表达。

我们构建了结核分枝杆菌H37Rv的hupB基因敲除突变株（H37Rv ΔhupB），并分别分析野生型H37Rv与该突变株在铁限制条件下的基因差异表达情况。实验中，我们分离了在高、低铁培养条件下生长的野生型H37Rv的RNA转录本，通过基因芯片（microarray）分析鉴定铁调控基因；同时对比分析铁限制条件下H37Rv ΔhupB与野生型H37Rv的基因表达差异。本次基因芯片检测由印度班加罗尔的安捷伦科技（Agilent Technologies）授权服务商Genotypic Technology完成商业化实验。

研究结果显示，低铁培养的野生型H37Rv中，参与分枝杆菌素生物合成的所有mbt基因，以及其他多种已报道的铁调控基因均出现显著上调。本研究的核心发现为：与低铁培养的野生型H37Rv相比，低铁培养的H37Rv ΔhupB无法实现mbt基因的上调表达。在受HupB调控的其他诸多基因中，该突变株的mmpL5与mmpS5转录本水平显著降低，这两个基因的表达产物据报道与铁载体的转运及生物合成密切相关。

本研究整体实验设计如下：采用单色芯片实验方案；实验生物为结核分枝杆菌；定制化结核分枝杆菌8×15k基因芯片由Genotypic Technology Private Limited设计（AMADID：20181）；样本标记使用安捷伦Quick-Amp标记试剂盒（货号：5190-0442）。